羟丁酸钠对缺氧缺血后新生大鼠海马CA1区Bcl-2、Bax蛋白表达的影响

目的：观察羟丁酸钠(GHB)对新生大鼠缺氧缺血性脑损伤（HIBD）后海马CA1区神经元Bcl-2、Bax蛋白表达的影响。方法：生后 7 d SD大鼠采用Rice等法，制成HIBD动物模型。新生大鼠随机分成假手术（sham）组、缺氧缺血（HI）组、GHB组。其中GHB组包括GHB50（50 mg/kg）、GHB100（100 mg/kg）、GHB200（200 mg/kg）亚组。各组在缺氧完成后 1 h、3 h、24 h、72 h 和 168 h 时点取脑切片作HE染色，用免疫组化染色观察Bcl-2、Bax蛋白的表达。结果：①光镜下HE染色结果：HI组海马CA1区锥体细胞排列紊乱，锥体细胞减少，海马带宽窄不一，可见细胞肿胀和核碎裂。GHB50组和GHB100组可减轻锥体细胞层病理改变。②免疫组化染色结果：HI组缺血缺氧后1h海马CA1区 Bcl-2、Bax表达开始增强，24 h 时达到高峰，其后逐渐减弱。在GHB50组和GHB100组可使Bcl-2表达明显高于HI组（P<0.05，P<0.01），Bax表达明显低于HI组（P<0.05，P<0.05）。结论：GHB可通过对Bcl-2、Bax表达的调控抑制新生大鼠HIBD后海马CA1区神经元损伤。     

CEPO调控新生大鼠缺氧缺血性脑损伤Bcl-2、Bax基因表达的研究

目的 观察新生大鼠缺氧缺血性脑损伤（HIBD）脑组织Bcl-2、Bax基因表达变化及氨基甲酰化促红细胞生成素（CEPO）干预对其表达的影响,探讨CEPO发挥脑保护作用的可能机制.方法 将新生7日龄SD大鼠建立HIBD模型,利用RT-PCR检测缺氧缺血和CEPO干预后2h、12h、24 h、48 h、72 h凋亡基因Bcl-2、Bax的mRNA表达的改变.结果 与假手术组相比,缺氧缺组在2h、12h、24 h、48 h、72 h脑组织中Bcl-2、Bax的表达均增加（P＜0.05）,与缺氧缺血组相比,CEPO干预组在不同时间点Bcl-2表达增加（P＜0.05）,Bax表达下降（P＜0.05）.结论 在新生大鼠HIBD中Bcl-2、Bax mRNA的表达发生改变,调控二者的表达水平可能是CEPO发挥脑保护的作用机制之一.     

脑缺氧缺血后新生大鼠海马CA1区NMDAR的表达

目的 :研究新生大鼠缺氧缺血性脑损伤 (HIBD)后 ,海马CA1区N甲基D天门冬氨酸受体 (NMDAR)及NMDARmRNA表达的变化。方法 :建立HIBD模型 ,用免疫组化及原位杂交方法 ,检测正常对照 (NORM)组和缺氧缺血 (HI)后不同时间点 ,NMDA受体I型亚单位 (NR1)表达的阳性细胞及NR1mRNA表达的阳性细胞。结果 :HI后 2h时NR1、NR1mR NA的表达稍下降 ,2 4h开始上升 ,72h达高峰 ,与正常对照组相比较有显著意义 (P <0 .0 5 )。结论 :正常新生大鼠海马CA1区有NR1及NR1mRNA的表达 ,HI后NR1及NR1mRNA的表达上调     

高压氧降低缺氧缺血性脑损伤大鼠脑组织内caspase-3表达

目的 探讨高压氧(HB0)对缺氧缺血新生大鼠神经细胞凋亡及easpase-3表达的影响.方法 建立缺氧缺血性脑损伤(HIBD)大鼠模型,将其分为HIBD组及HBO处理组,并设立假手术组(n=24).观察3组大鼠不同时间脑组织病理学改变,同时用免疫组化的方法检测3组大鼠脑组织内ca8pase-3蛋白表达的动态变化.结果 HIBD组18、24、48和96 h时间点海马及皮层区ca8pase-3蛋白的表达均明显高于假手术组(P相似文献   

米诺环素对缺氧缺血脑损伤未成熟新生大鼠TLR4、NF—κBp65和TNF—α表达的影响

目的观察米诺环素（Minocycline,MN）对缺氧缺血脑损伤（hypoxic-ischemic brain damage,HIBD）未成熟新生大鼠Toll样受体4（Toll-1ike receptor4,TLR4）、核因子-κB（nuclear factor-kappa B,NF—κB）p65和TNF．仪表达的影响,探索米诺环素脑保护作用机制。方法将160只生后2d（P2）Sprague—Dawley（SD）新生大鼠随机分成正常对照组、假手术组、HIBD组、HIBD加MN组。通过结扎左侧颈总动脉及8％氮氧混合气缺氧4h,制备未成熟新生大鼠缺氧缺血性脑损伤模型。HIBD加MN组大鼠缺氧后予腹腔注射1次MN45mg／kg,HIBD组予腹腔注射等剂量的无菌PBS（pH7．4）。HI后24h、48h、72h取材,Westernblotting检测TLR4、NF．KBp65和TNF—Ot蛋白表达,HI后72h、4周行脑组织HE染色及病理评分,HI后4周行行为学检测。结果HI后72h、4周HIBD加MN组脑组织病理损伤较HIBD组减轻,HIBD加MN组半定量病理评分低于HIBD组,差异有统计学意义（P〈0．05）。Western blotting显示HI后24、48和72hHIBD加MN组TLR4、NF—KBp65和TNF-α的表达低于HIBD组,较正常组及假手术组升高。HI后4周,在悬吊试验及斜坡试验中,HIBD加MN组与正常组、假手术组差异无统计学意义（Jp〉0．05）。旷场试验中,HIBD加MN组与正常组、假手术组对比,差异有统计学意义（P〈0．05）;与HIBD组比较,P=0．375,差异无统计学意义（P〉O．05）。圆筒实验中HIBD加MN组左侧上肢触壁百分比较HIBD组降低（P〈0．05）,与正常组、假手术组差异无统计学意义（P〉0．05）;右侧触壁百分比的比较中,HIBD加MN组与正常组、假手术组、HIBD组差异无统计学意义（P〉0．05）。结论米诺环素对缺氧缺血脑损伤近期及远期具有良好的保护作用,其对缺氧缺血脑损伤的保护作用机制可能与抑制TLR4．NF—κBp65-TNF-α信号途径的激活有关。     

新生鼠缺氧缺血脑损伤TGF-β1表达与神经细胞凋亡

目的 研究新生大鼠脑缺氧缺血（HI）后TGF－β1表达和神经细胞凋亡的变化规律，探讨新生儿缺氧缺血脑损伤的发病机制。方法 结扎新生7d龄SD大鼠左颈总动脉后，吸入8％浓度氧2h，建立HIBD模型。应用苏木素－伊红（HE）染色，原位缺口末端标记（TUNEL）及SP免疫组化方法检测新生大鼠HI后存活不同时间大脑皮质和民TGF－β1的表达及神经细胞凋亡的情况。结果 缺氧缺血后8h，大脑皮质和海马出现TGF－β1的表达，缺氧缺血后12h和48h ,TGF－β1出现两次表达高峰，神经细胞凋亡高峰为缺氧缺血后24h，晚期表达TGF－β1的免疫阳性细胞与凋亡细胞均出现在缺血半暗带内。结论 缺氧缺血引起了TGF－β1的表达增强，TGF－β1的表达可能通过对神经细胞凋亡的调控，参与缺氧缺血后神经细胞的修复。     

木犀草素对缺氧缺血性脑损伤新生大鼠的神经保护作用

目的：探究木犀草素（Lut）对缺氧缺血性脑损伤（HIBD）新生大鼠是否具有神经保护作用及其作用机制。方法：将7日龄新生大鼠分为假手术组、模型组（HIBD组）和Lut治疗组（HIBD+Lut组）。用Rice-Vannucci法建立新生大鼠HIBD模型。HIBD+Lut组在造模后即刻通过腹腔注射给予50 mg/kg Lut,连续3 d,模型组和假手术组同时腹腔注射等体积生理盐水。3 d后,用氯化三苯基四氮唑（TTC）染色评估脑梗死范围;用干/湿重脑含水量法评估缺血脑半球的脑水肿情况;用苏木精-伊红（HE）染色以及原位末端转移酶标记（TUNEL）和神经元核抗原（NeuN）荧光双标共定位法观察缺血脑半球海马和皮质中神经元损伤情况;用商用试剂盒检测缺血脑半球中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）活性及丙二醛（MDA）水平;用Western blot法检测缺血脑半球的海马和皮质中核因子E2相关因子2(NRF-2)和血红素加氧酶1(HO-1)的蛋白表达水平。取35日龄大鼠,用水迷宫实验评估大鼠的认知功能情况。结果：与假手术组比较,HIBD组大鼠脑梗死、脑...     

新生大鼠缺氧缺血性脑损伤caspase-3表达与自由基损伤机制的研究

目的:研究缺氧缺血性脑损伤(HIBD)新生大鼠caspase-3表达与自由基损伤的机制。方法:建立7日龄新生大鼠HIBD模型,分为HIBD组和假手术组,两组分别于HI后6、12、24、48、72、96 h取脑组织,应用TUNEL法检测神经细胞凋亡,免疫组织化学法检测caspase-3蛋白的表达,硫代巴比妥酸法测定丙二醛(MDA)含量,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)含量。结果:HIBD组神经细胞凋亡数量及caspase-3的表达量6 h开始增多,48 h达高峰,各时间点两者均显著高于假手术组(P0.05)。HIBD组MDA含量6 h增高,24 h达高峰,各时间点均较假手术组增高(P0.05)。HIBD组SOD含量6 h下降,24 h降至最低,各时间点均较假手术组降低(P0.05)。凋亡细胞数量与caspase-3蛋白表达量和MDA含量均呈正相关(r=0.748、0.654,P均0.05),与SOD含量呈负相关(r=-0.576,P0.05)。结论:HIBD时自由基损伤促进caspase-3的表达及神经细胞的凋亡。     

舒芬太尼调节cAMP/PKA/CREB通路减轻HIBD新生大鼠神经元损伤

目的 探讨舒芬太尼通过调节环磷酸腺苷（cAMP）/蛋白激酶A(PKA)/cAMP反应元件结合蛋白（CREB信号通路减轻缺氧缺血性脑损伤（HIBD）新生大鼠神经元损伤的机制。方法 采用Rice-Vannucci法构建HIBD新生大鼠模型，随机分为：模型组、舒芬太尼低剂量组、舒芬太尼高剂量组、SQ22536组、舒芬太尼高剂量+SQ22536组，每组10只，另取10只新生大鼠设为假手术组，以舒芬太尼和SQ22536分组干预后，以避暗实验检测大鼠认知功能；以伊文思蓝实验检测大鼠血脑屏障功能；以H-E染色检测各组大鼠脑皮质与海马神经元病理形态；以尼氏染色检测各组大鼠脑皮质与海马神经元数量；以试剂盒检测各组大鼠血清炎性因子前列腺素2(PGE2)、诱导型一氧化氮合酶（iNOS）、抗氧化因子总抗氧化能力（TAC）、超氧化物歧化酶（SOD）水平及脑组织cAMP水平；以免疫印迹法检测各组大鼠脑组织cAMP/PKA/CREB通路相关表达。结果 与假手术组比较，模型组大鼠脑皮质与海马神经元均病理损伤严重，步入潜伏期、脑皮质与海马神经元数量、血清TAC与SOD水平、脑组织cAMP水平及p-PKA/PKA、p-C...     

缺氧缺血脑损伤大鼠脑组织Cofilin 1与ERK1/2蛋白表达与磷酸化的分析

目的探索新生大鼠缺氧缺血脑损伤模型脑组织Cofilin1与ERK1/2蛋白表达与磷酸化及其在脑组织中分布的规律。方法新生7日龄SD大鼠,手术结扎左侧颈总动脉并经低氧处理造成缺氧缺血脑损伤(HIBD)模型,以假手术组为对照,应用western Blot和免疫组化方法检测损伤后一定时间段(0h～48h)损伤侧脑组织Cofilin1与磷酸化cofilin1(p-Cofilin1)及Erk1/2和磷酸化ERK1/2(p-ERK1/2)的表达变化,同时检测其在损伤大鼠脑组织和细胞中的表达和分布。结果 Western blot检测显示,HIBD新生SD大鼠脑组织中Cofilin1在HI后的表达与对照组相比也未见明显改变,但磷酸化Cofilin1在HI后1h～12h之间降低,24h后恢复正常;ERK1/2蛋白在不同时点的表达与对照组相比未见明显差异,但可见磷酸化ERK1/2(p-ERK1/2)在缺氧缺血处理(HI)后0h略低于正常,而在1-2h内迅速升高并高于正常对照和假手术,在2h后又逐渐降低。免疫组化结果显示Cofilin1与p-cofilin1在损伤大鼠脑中主要分布于海马和大脑皮质,尤其p-Cofilin表达分布以海马的颗粒细胞为主,并且在HI后7d-14d表达明显增强,在21d则明显降低。结论 Cofilin和ERK的磷酸化修饰与缺氧缺血脑损伤的发生和发展过程有较密切的关系,且cofilin1和p-cofilin1在大脑海马区的定位分布,提示其可能与大脑的学习记忆功能的损伤与修复有一定的关系。     

新生大鼠缺氧缺血性损伤致脑白质自噬体形成增多*

目的：探讨缺氧缺血对新生大鼠脑白质自噬体形成及髓鞘生成的影响。方法： 3 日龄的SD大鼠随机分为 假手术组（ sham）和缺氧组（ HI）,每组又分为24 h、48 h、72 h、5 d、7 d 5 个时间点,透射电镜观察脑白质自 噬发生情况及髓鞘生成情况,免疫印迹检测自噬相关蛋白LC3和髓鞘碱性蛋白（ MBP）的表达情况。结果： HI 24 h 组自噬体增多,散在分布;HI 48 h 组自噬体增多更明显,较多细胞突起内可见成堆聚集的自噬体;HI 72 h 组自噬体和HI 48 h 组比较略少,与sham 组比较差异均有统计学意义。MBP表达下调,其中HI 7 d 组与sham 组 比较,差异有统计学意义, HI 7 d 组髓鞘形成疏松,板层结构紊乱。结论： 新生大鼠脑缺氧缺血可诱导脑白质发 生自噬,在缺氧缺血后48 h 达到高峰;新生大鼠缺氧缺血后不仅MBP合成减少,新生髓鞘结构也异常。     

过表达Olig2的少突胶质前体细胞在缺血缺氧脑白质损伤新生大鼠脑内的分化

目的:观察过表达特异性转录因子Olig2的少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)移植在缺血缺氧(hypoxia-ischemia,HI)脑白质损伤新生大鼠脑内的分化情况。方法:用绿色荧光蛋白(green fluorescent protein,GFP)标记的过表达Olig2的慢病毒感染原代分离纯化的大鼠大脑皮层OPCs,GFP阳性细胞计数测其感染率。将过表达Olig2的OPCs(Olig2组)或阴性对照病毒感染的OPCs(Vector组)脑立体定位注射到造模后7 d的HI模型大鼠胼胝体膝部,移植后2周,冰冻切片行免疫荧光染色观察OPCs的存活和分化情况。结果:Olig2-GV218病毒感染OPCs细胞48 h,荧光显微镜检测显示85%左右的OPCs细胞表达GFP;移植后2周,caspase-3荧光染色表明移植后绝大部分细胞存活,Vector组和Olig2组之间无统计学差异(P0.05);GFAP/GFP双阳性细胞在两组之间也无显著差异(P0.05);而Olig2组MBP/GFP双阳性细胞的荧光密度显著高于Vector组(P0.05)。结论:过表达Olig2可促进移植OPCs向少突胶质细胞分化。     

JNK通路促进大鼠脑缺血再灌注海马神经元凋亡

目的:探讨c-JunN端激酶(JNK)通路在大鼠脑缺血再灌注后海马神经元凋亡中的作用。方法:雄性SD大鼠90只,随机分为假手术组、全脑缺血再灌注组、全脑缺血再灌注+JNK抑制剂(SP600125)组、全脑缺血再灌注+JNK激动剂(茴香霉素)组和全脑缺血再灌注+溶剂对照组,每组再灌注后24h取材。分别采用免疫组化、Westernblotting和实时荧光定量PCR检测海马神经元caspase-3蛋白和mRNA的表达;采用TUNEL染色检测海马神经元凋亡情况。结果:全脑缺血再灌注组caspase-3蛋白和mRNA表达较假手术组增加(P<0.05);与全脑缺血再灌注组相比,全脑缺血再灌注+JNK抑制剂组caspase-3蛋白和mRNA表达均降低(P<0.05),而全脑缺血再灌注+JNK激动剂组caspase-3蛋白和mRNA表达均增加(P<0.05),全脑缺血再灌注+溶剂对照组则无明显变化(P>0.05)。各组海马神经元凋亡趋势与caspase-3蛋白和mRNA变化趋势一致。结论:JNK通路的激活可增加大鼠脑缺血再灌注后海马神经元caspase-3的表达,促进海马神经元凋亡。     

新生大鼠低氧缺血脑损伤后c-fos在脑神经元中的表达

目的：探讨c-fos基因表达在新生大鼠脑低氧缺血损伤中所起的作用。方法：采用免疫组化及逆转录扩增方法,观察新生大鼠脑低氧缺血后皮质、海马组织中c-fos基因转录、翻译水平的表达现象。结果：脑低氧缺血后早期海马和皮质部位可同时诱导出c-fos mRNA和c-fos蛋白（与sham组比较P<0.05）;皮质表达的c-fos明显低于海马（P<0.05）,非结扎侧c-fos基因一过性表达增高,但与结扎侧比较有明显差异（P<0.05）。结论：新生大鼠脑组织在低氧缺血后具备能产生特殊基因表达改变的功能,其c-fos基因的表达可能对于脑损伤后细胞的恢复与生存有重要意义。     

Failure to complete apoptosis following neonatal hypoxia-ischemia manifests as "continuum" phenotype of cell death and occurs with multiple manifestations of mitochondrial dysfunction in rodent forebrain

Controversy surrounds proper classification of neurodegeneration occurring acutely following neonatal hypoxia-ischemia (HI). By ultrastructural classification, in the first 24 h after neonatal hypoxia-ischemia in the 7-day-old (p7) rat, the majority of striatal cells die having both apoptotic and necrotic features. There is formation of a functional apoptosome, and activation of caspases-9 and -3 occurring simultaneously with loss of structurally intact mitochondria to 34.7+/-25% and loss of mitochondrial cytochrome c oxidase activity to 34.7+/-12.7% of control levels by 3 h after hypoxia-ischemia. There is also loss of the mitochondrial motor protein, kinesin. This combination of activation of apoptosis pathways simultaneous with significant mitochondrial dysfunction may cause incomplete packaging of nuclear and cytoplasmic contents and a hybrid of necrotic and apoptotic features. Evidence for an intermediate biochemistry of cell death including expression of the 17 kDa isoform of caspase-3 in dying neurons lacking a classic apoptotic morphology and degradation of the neuronal cytoskeletal protein spectrin by caspase-3 and calcium-activated calpains yielding 120 kDa and 145/150 kDa fragments, respectively, is also found. In summary, neonatal hypoxia-ischemia triggers apoptotic cascades, and simultaneously causes mitochondrial structural and functional failure. The presence of a "continuum" phenotype of cell death that varies on a cell-by-cell basis suggests that the phenotype of cell death is dependent on the energy available to drive the apoptotic pathways to completion.     

Effect of sodium hydroxybutyrate on the expression of hippocampal N-methyl-D-aspartate receptor 2B subunit mRNA in neonatal rats with hypoxic-ischemic insult

The objective of this research was to test whether sodium hydroxybutyrate (GHB-Na) protects rat neonatal brain against hypoxia-ischemia (HI). Specifically, the objective was to determine the effect of GHB-Na administration on the expression of N-methyl-D-aspartate subunit (NR2B) mRNA in the rat hippocampus. Seven-day-old Sprague-Dawley rats were subjected to ligation of the left carotid artery and were randomly assigned to 5 groups: sham operated (S), saline treated (C), and those treated with GHB-Na (G1, G2, G3), at 3 dosages (50, 100, or 200 mg/kg, ip, thrice daily). NR2B mRNA levels in the left hippocampus were assayed at 2, 6, 12, 24, 72, and 168 hr after HI (exposure to 8% O(2)/92% N atmosphere for 2 hr). The results suggest that HI insult increased NR2B mRNA gene expression in the left hippocampus of the neonatal rats and that GHB-Na administration partially suppressed this effect of HI insult.     

Granulocyte stimulating factor attenuates hypoxic-ischemic brain injury by inhibiting apoptosis in neonatal rats

Purpose

This study was undertaken to determine the neuroprotective effect of granulocyte stimulating factor (G-CSF) on neonatal hypoxic-ischemic brain injury.

Materials and Methods

Seven-day-old male newborn rat pups were subjected to 110 minutes of 8% oxygen following a unilateral carotid artery ligation. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluorescinated annexin V and propidium iodide (PI) and JC-1 (5,5'',6,6''-tetrachloro-1,1'',3,3''-tetraethylbenzimidazolyl-carbocyanine iodide). The extent of cerebral infarction was evaluated at 2 weeks after recovery.

Results

With a single dose (50 µg/ kg) of G-CSF treatment immediately after hypoxic-ischemic insult, hypoxia-ischemia induced increase in TUNEL-positive cells, annexinV+/PI-and JC-1 positive apoptotic cells in the ipsilateral cerebral cortex was significantly reduced at 24 hours, measured by flow cytometry, and the extent of cerebral infarction at 2 weeks after recovery was also significantly attenuated compared to the hypoxia-ischemia control group.

Conclusion

Our data suggest that G-CSF is neuroprotective by inhibiting apoptosis, thereby reducing the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia (HI).     

Effects of endotoxin administration and cerebral hypoxia-ischemia on complement activity and local transcriptional regulation in neonatal rats

It is not known whether up-regulation of complement components, either circulating or locally synthesized, contributes to an increased susceptibility to neonatal hypoxic-ischemic (HI) cerebral injury. Therefore, we tested the hypothesis that in neonatal rats subjected to a unilateral HI cerebral insult, prior administration of E. coli lipopolysaccharide (LPS) augments (1) complement-mediated serum hemolytic activity, and (2) C3 mRNA and C9 mRNA levels in hepatic and cerebral tissue. Pregnant rats were injected subcutaneously with sterile normal saline (NS) or 500 microg/kg of LPS on gestational days 18 and 19. Following birth, the pups received intraperitoneal injections of NS or 250 microg/kg of LPS on postnatal days 3 and 5. On postnatal day 7, each animal was subjected to ligation of the right common carotid artery followed by 2.5h of hypoxia (8% O(2)). At 3, 6,18, 24 and 48 h after hypoxia, the complement-mediated hemolytic activity of pooled serum was measured. Hepatic and cerebral C3 mRNA and C9 mRNA were quantified by qRT-PCR at 3, 6, and 18 h after HI. Serum hemolytic activity, hepatic C3 mRNA, and hepatic C9 mRNA were up-regulated after cerebral HI. LPS administration potentiated the effect of HI on serum hemolytic activity and increased cerebral C3 mRNA levels. Cerebral C9 mRNA was not detected and was not affected by HI, with or without the prior LPS administration. These observations support the theory that previously reported C9-mediated neurotoxicity following cerebral HI is induced by circulating, rather than locally synthesized C9.